Parkinson's disease (PD) is a common neurological syndrome characterized by the selective loss of dopaminergic neurons in the nigrostriatal tract. Specifically, dopamine neurons in the substantia nigra degenerate, resulting in the loss of dopamine (3,4-dihydroxyphenethylamine) input to the striatum. Clinically, the reduction of dopamine in the striatum causes several symptoms such as increased muscle rigidity, resting tremor, bradykinesia, and abnormalities of posture and gait. The level of decrease in dopamine synthesis correlates with the severity of the symptoms. Without treatment, PD patients eventually progress to a tragically debilitating rigid state.
Current treatment regimes for PD consist primarily of pharmacological supplementation of the dopaminergic loss with dopamine agonists and levodopa (3-hydroxy-L-tyrosine, L-DOPA), the metabolic precursor of dopamine, which, unlike dopamine, can readily cross the blood-brain barrier (for a general review of PD treatments currently in use see Adams et al., Principles of Neurology 4.sup.th Ed. McGraw Hill, New York (1989). However, conventional treatments for Parkinson's disease with L-DOPA have proven to be inadequate for many reasons of record in the medical literature. The systemic administration of levodopa, although producing clinically beneficial effects at first, is complicated by the need to reduce dosages that were well tolerated at the outset in order to avoid side effects.
The reason that adverse effects develop in this way is unclear, but selective denervation or drug-induced supersensitivity may be responsible. Some patients also become less responsive to levodopa, so that previously effective doses eventually fail to produce any therapeutic benefit. It is not clear whether this relates to disease progression or to duration of treatment, although the evidence is increasing that disease progression is primarily responsible for the declining response. Responsiveness to levodopa may ultimately be lost completely, perhaps because of the disappearance of dopaminergic nigrostriatal nerve terminals or some pathologic process directly involving the striatal dopamine receptors. For such reasons, the benefits of levodopa treatment often begin to diminish after about 3 or 4 years of therapy irrespective of the initial therapeutic response. In addition, the augmentation of systemic levels of levodopa, necessary to establish therapeutically effective levels at the site of interest, i.e., the brain, have been reported to cause several gastrointestinal adverse effects (including anorexia, nausea and vomiting due to the stimulation of an emetic center located in the brain stem outside the blood-brain barrier), cardiovascular effects (mostly due to the increased catecholamine formation peripherally), dyskinesia, and drastic behavioral effects (depression, anxiety, agitation, insomnia, somnolence, confusion, delusions, hallucinations, psychotic episodes and other changes in mood or personality).
The peripheral administration of levodopa is further complicated by the fact that only about 1-3% of administered levodopa actually enters the brain unaltered, the remainder being metabolized extracerebrally, predominantly by decarboxylation to dopamine, which does not penetrate the blood-brain barrier. This means that levodopa must be given in large amounts when it is used alone. The co-administration of a peripheral dopadecarboxylase has been found to reduce the dosage requirements and some of the side effects, although only marginally. Finally, certain fluctuations in clinical response to levodopa occur with increasing frequency as treatment continues. In some patients, these fluctuations relate to the timing of levodopa intake, and they are then referred to as wearing-off reactions or end-of-dose akinesia. In other instances, fluctuations in clinical state are unrelated to the timing of doses (on-off phenomenon). In the on-off phenomenon, off-periods of marked akinesia alternate over the course of a few hours with on-periods of improved mobility but often marked dyskinesia. (Aminoff, "Parkinson's Disease and other Extrapyramidal Disorders", in Harrison's Principles of Internal Medicine, 14.sup.th Ed. McGraw-Hill, (1998), pp. 2356-2359), and Katzung Basic & Clinical Pharmacology, 6.sup.th Ed., Appleton & Lange, Norwalk, Conn.) Thus, there is currently no clearly effective cure for PD.
Since 1987, investigators have grafted primary human fetal cells attempting to supplement nigrostriatal neurons to affected patients. Hoffer et al., (1991) Trends Neurosci. 14:384-388) reports that this approach is replete with obstacles including variability in clinical recovery post transplantation, quality control for viral and retroviral contamination problems, nonrenewable sources, and most of all ethical and moral obstacles.
Several laboratories have sought to formulate more effective PD treatment regimens by manipulating a cofactor essential to the activity of tyrosine hydroxylase (TH). Levine et al., (1981) Science 214:919-921) teaches that (6R)-5,6,7,8-tetrahydro-L-biopterin (BH.sub.4) is localized in dopaminergic nerve terminals in the striatum. The role of BH.sub.4 in biogenic amine neurotransmitter metabolism has been extensively studied. As the cofactor for tyrosine and tryptophan hydroxylases, BH.sub.4 has been postulated to play a pivotal role in the regulation of biogenic neurotransmitter biosynthesis (see, Levine et al., in Biochemical and Clinical Aspects of Pteridines, Vol. 2, pages 325-337, Walter de Gruyter, Berlin (1983) and Nagatsu et al., (1996) Neur. Res. 21(2):245-250). BH.sub.4 deficiency has been correlated with several diseases including Parkinson's disease (PD) (Lovenberg et al., (1979) Science 204:624-626), Alzheimer's disease (Williams et al., (1980) Psychiatry 43:735-738), and familial Dystonia (Williams et al., (1979) Lancet 2:410-411. Unfortunately, these earlier attempts failed. Le Witt et al., (1986) Neurology 36:760-764, teaches that the main obstacle to the development of therapeutic approaches is that BH.sub.4 does not readily cross the blood-brain barrier, and thus peripheral supplementation fails to result in therapeutically effective BH.sub.4 concentrations in the cerebral pools.
Gene delivery has been attempted to provide functional copies of the cDNAs encoding proteins necessary for the augmentation of dopamine synthesis. For example, several ex vivo studies have been done using tumorigenic or primary cells expressing rat or human TH. These studies report performing genetic modifications that result in elevated TH gene expression (see for example, PCT Publications WO 96/39496, and WO 98/18934). However, it appears that cells used as vehicles in these studies were consistently deficient in sufficient levels of BH.sub.4 cofactor for TH activity. This could be seen in experiments where production of L-DOPA from TH-producing cells in culture was dependent on addition of micromolar to millimolar concentrations of BH.sub.4 to the media.
Attempts to augment the production of L-DOPA in TH-producing cells led to the development of methods including the supplementation of BH.sub.4. Uchida et al., (1990) Neuroscience Letters 109:282-286, and Ishida et al., (1996) Cell Transplantation 5:S5-S7, disclose that fibroblasts transfected with various constructs expressing a TH cDNA were able to produce L-DOPA only in the presence of BH.sub.4 in the medium. Uchida et al., (supra), and Uchida et al. (1992) Dev. Neurosci. 14:173-180 reported that administration of BH.sub.4 through a microdialysis probe greatly enhances or enables measurement of in vivo L-DOPA, DA and metabolites in intracerebrally grafted fibroblasts. Unfortunately, these in vivo experiments do not have therapeutic applications since the direct administration of BH.sub.4 to the brain is clinically impossible
More recently, efforts have been directed to the introduction of both GTP cyclohydrolase, an enzyme critical for BH.sub.4 synthesis, and TH to supply both BH.sub.4 and TH. Bencsics et al., (1996) J. Neurosci. 16(14):4449-4456) teaches the production of dopamine in co-transfected fibroblasts. Similarly, PCT publication WO 96/05319 discloses the transfection of two or more constructs to provide BH.sub.4 as well as TH. More recently, Mandel et al., (1998) J. Neurosci. 18(11):4271-4284 reported in vivo production of dopamine in a rat model of PD by intrastriatal adeno-associated virus gene transfer of GTP cyclohydrolase and TH (see also PCT publication WO 97/1831). Unfortunately, despite these recent successes, from a clinical perspective much work remains to be done. In a clinical setting, it is often necessary to modulate the production of L-DOPA and dopamine to address a particular patient's requirements, as is the case with any pharmacologic treatment. Furthermore, clinical requirements include the need to target specific tissues to the exclusion of others to avoid the serious side effects discussed above. None of these systems allows the external modulation of the level of L-DOPA and of dopamine expression. Moreover, none of the currently available methodologies provide protocols achieving localized L-DOPA production. The coordination of drug delivery with gene therapy as a treatment, which must also meet both clinical and FDA safety parameters, adds an entire novel dimension to the applicability of this work.
Thus, a great need exists for more effective and versatile gene delivery approaches to modulate striatal L-DOPA and/or dopamine production in a clinically safer and therapeutically effective fashion. Such methods should allow the manipulation of TH activity to address each patient's requirements, such as the stage of neuronal degeneration, the age and condition of the patient, interactions with other medications taken by the patient, side-effects, and the like. It is to this end that this invention is directed.